The Nck-interacting kinase phosphorylates ERM proteins for formation of lamellipodium by growth factors

Abstract

The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. We report two previously unrecognized actions of NIK: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressing a kinase-inactive NIK, suppressed for NIK expression with siRNA oligonucleotides, or expressing ezrin T567A that cannot be phosphorylated. These data suggest that direct phosphorylation of ERM proteins by NIK constitutes a signaling mechanism controlling growth factor-induced membrane protrusion and cell morphology.ezrin ͉ moesin ͉ ste20 kinase ͉ membrane protrusion T he Nck-interacting kinase (NIK) is a member of the germinal center kinase subfamily of Ste20͞MAP4K serine͞threonine kinases (1). Closely related to NIK are the mammalian kinases TNIK (2), MINK (3), and NRK͞NESK (4) and orthologs Misshapen (Msn) in Drosophila (5) and MIG-15 in Caenorhabditis elegans (6). NIK and its orthologs share a common function in regulating cell shape and migration. In mice, homozygous knockout of NIK results in early embryonic lethality with defects in mesoderm migration (7), and expression of kinase-inactive NIK attenuates epithelial cell invasion (8). Msn functions in determining epithelial polarity, dorsal closure. and neuronal targeting (5, 9, 10), and MIG-15 controls axonal navigation (6). NIK (1) and Msn (5) also share a conserved activation of the JNK pathway. NIK, however, does not directly phosphorylate JNK, nor do NIK nullizygous embryos precisely phenocopy mice lacking JNK1 or JNK2 (7). Additionally, activation of JNK is not associated with dynamic changes in cell morphology. Hence, NIK substrates that control cell morphogenesis have not been identified.We now report that the ERM proteins ezrin, radixin, and moesin are substrates for NIK. ERM proteins regulate cell morphology by cross-linking actin filaments to the plasma membrane. The N-terminal FERM (4.1 ERM) domain of ERM proteins binds to integral plasma membrane proteins and the C terminus binds F-actin (11). ERM proteins control cell shape primarily by regulating membrane protrusions and cell-substrate adhesion. In epithelial cells ERM proteins are necessary for the formation of apical microvilli (12)(13)(14). In fibroblasts ERM proteins regulate the assembly of focal adhesions (...

LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation

“…HGC was reported to phosphorylate ERM in the lamellipodium of growth factor-stimulated human epithelial cells (19). Drosophila provides a particularly informative model because of its reduced number of kinases and a single ERM protein.…”

“…Second, studies in the important model organism Drosophila have implicated a kinase called SLIK, from a very divergent family of kinases, the STE20-related family (STE) (5,6,18). More recently one mammalian kinase in the STE family, HGK, was proposed to function as an ERM kinase (19). Thus, mammalian ERM kinases likely include ones outside the AGC family and in the STE family.…”

Activated Ezrin Promotes Cell Migration through Recruitment of the GEF Dbl to Lipid Rafts and Preferential Downstream Activation of Cdc42

“…The T567D form rescues because it does not require the activation machinery, i.e., it is already in the open conformation to bind to Dbl at the right location, where the membrane is locally tethered to the cytoskeleton. Potential N-ERMAD-interacting partners that contribute to the activation process include the various protein kinases that phosphorylate the conserved C-terminal threonine (myotonic dystrophy kinaserelated Cdc42-binding kinase [Nakamura et al, 2000], protein kinase C␣ [Ng et al, 2001], and Nck-interacting kinase [Baumgartner et al, 2006]) as well as the phosphoinositide PIP 2 (Fievet et al, 2004). PIP 2 (IP 3 head) binding was shown to require several lysine residues from subdomains A and C of N-ERMAD, which form the positively charged molecular surface to interact with the negatively charged membrane inositol polyphosphates and, in the absence of PIP 2 , moesin does not bind CD44 in vitro (Hamada et al, 2000).…”

“…This sequence of molecular events releases the intramolecular interaction between the N-and C-termini. Several kinases, including myotonic dystrophy kinase-related Cdc42-binding kinase Several kinases, including myotonic dystrophy kinase-related Cdc42-binding kinase (Nakamura et al, 2000), protein kinase C␣ (Ng et al, 2001), and Nck-interacting kinase (Baumgartner et al, 2006) have been shown to phosphorylate the conserved C-terminal threonine in ERM proteins. This second activation step of phosphorylation may restrict actin tethering by ERM proteins to specialized membrane domains, such as the protruding cell membranes, by locally unmasking the binding site for actin filaments in C-ERMAD.…”

Model membranes to shed light on the biochemical and physical properties of ezrin/radixin/moesin

“…For example, in cellulo, moesin is phosphorylated by Rho kinase during platelet activation [53,54] or by lymphocyte-oriented kinase (LOK) which stimulates lymphocyte migration and polarization [55]. A number of other kinases in vertebrate cells can phosphorylate ERMs on this regulatory threonine, including protein kinase C (PKC) α and θ, Nck-interacting kinase (NIK), human serine/threonine kinase (Mst4) [56][57][58][59]. Phosphorylation of ERM proteins resulted in the localization of these proteins to the actin-rich membrane extensions [53,54,60,61].…”